Endothelial to Mesenchymal Transition (EndMT) plays a key role in embryonic development and in adult cardiovascular disease (CVD). While our team is at the forefront in defining the importance of EndMT in CVD, our overall knowledge of this process remains in its infancy. The broad objective of this R01 is to leverage novel discoveries made by our group to define the core mechanisms of EndMT in adult CVD, with an ultimate view to clinical translation. Aim 1 will define the causal role of Zeb2 in EndMT and atherosclerotic disease. We have identified a likely biologic intersection point between CAD, EndMT and the gene Zeb2. Zeb2 is known to modulate EndMT-related cellular processes, while a Zeb2-associated polymorphism (rs2252641) is one of only 46 independent CAD risk alleles. We further validated the importance of Zeb2 in CAD using our unique STARNET dataset, a genetics-of-gene expression study involving 1062 patients with and without CAD. In Aim 1 we will combine in vitro studies of Zeb2 knockdown during EndMT, in vivo rodent studies of tissue- specific Zeb2 knockdown in an atherosclerosis model, and systems genetics studies of Zeb2 using the STARNET dataset. We anticipate that Aim 1 will make major inroads on our mechanistic understanding of Zeb2, its effect in promoting CAD and its role in EndMT. Given that Zeb2 is a CAD risk allele but yet its function in this disease is unknown, this Aim is of significant clinical importance. Aim 2 will define the effect of EndMT inhibition in a large animal model of vein graft remodeling and obtain preclinical proof-of- principle data for this approach. We have shown that EndMT is critical during vein graft remodeling when sections of vein are exposed to arterial pressure. Antagonism of endothelial TGF-? signaling in mice by altered Smad2/3-Snai2 signaling led to reduced EndMT during vein graft remodeling and less neointimal formation. In Aim 2 we will generate adeno-associated viral vector (AAV) 1 constructs targeting Smad3 and Snai2 and apply these to an established arterio-venous fistula porcine model to define the effect of EndMT inhibition on vein graft remodeling. We expect Aim 2 to provide definitive data as to the possibility of inhibiting EndMT to improve vein graft remodeling such that grafts/fistulae have reduced neointima, increased luminal size and ultimately, improved patency. Aim 3 will define key drivers and regulators of EndMT in the adult. Using our unique in vitro EndMT model we will drive disease-relevant endothelial cells toward a mesenchymal phenotype and conduct in-depth studies of the transcriptional and epigenetic regulators of EndMT. Systems genetics will be used to integrate these transcriptional, epigenetic and other regulators of EndMT to define key hierarchical drivers of EndMT. We expect that Aim 3 will greatly enrich our understanding of the core molecular and epigenetic mechanisms of EndMT. Collectively, this project will shift current paradigms and firmly establish the role of EndMT, and the therapeutic opportunities for EndMT manipulation, in adult CVD.